The long term aim of this project is to understand the role of RNA processing in gene expression and animal development. One of the main problems in studying eukaryotic mRNA biogenesis has been isolating the RNA substrate for processing reactions, namely pre-mRNA. This problem has been circumvented by employing an efficient prokaryotic RNA polymerase and promoter to synthesize large amounts of eukaryotic pre-mRNAs in vitro. Most importantly, these in vitro synthesized pre-mRNAs are accurately processed (spliced) following injection into frog oocyte nuclei. The in vitro transcription-oocyte injection system makes it possible to design and synthesize mutant mRNAs and pre-mRNAs and to test their biological function in a living cell. This experimental design will be used to identify and characterize RNA sequences important for RNA splicing, transport, and 3' end formation. In addition, special attention will be given to analyzing how RNAs are localized to particular regions of egg cytoplasm. Several cloned eukaryotic genes, including globin genes, histone genes, and genes encoding maternal mRNAs, will be used to study these processing events. As an extension of the in vivo assays, an in vitro RNA processing system will be established using isolated oocyte nuclei. If successful, these in vitro studies will significantly advance the understanding of the biochemical mechanisms of RNA processing, particularly splicing. The proposed experiments will lead to a better understanding of how animal cells synthesize and regulate the use of messenger RNAs. This basic information may be important for understanding how RNA processing defects cause cellular dysfunction as in the case of some human thalassemias.